Schultheiß et al. BMC Evolutionary Biology 2014, 14:42 http://www.biomedcentral.com/1471-2148/14/42

RESEARCH ARTICLE Open Access Disjunct distributions of freshwater snails testify to a central role of the Congo system in shaping biogeographical patterns in Africa Roland Schultheiß1,2*, Bert Van Bocxlaer3,4, Frank Riedel5,6, Thomas von Rintelen7 and Christian Albrecht2

Abstract Background: The formation of the East African Rift System has decisively influenced the distribution and evolution of tropical Africa’s biota by altering climate conditions, by creating basins for large long-lived lakes, and by affecting the catchment and drainage directions of river systems. However, it remains unclear how rifting affected the biogeographical patterns of freshwater biota through time on a continental scale, which is further complicated by the scarcity of molecular data from the largest African river system, the Congo. Results: We study these biogeographical patterns using a fossil-calibrated multi-locus phylogeny of the gastropod family . This group allows reconstructing drainage patterns exceptionally well because it disperses very poorly in the absence of existing freshwater connections. Our phylogeny covers localities from major drainage basins of tropical Africa and reveals highly disjunct sister-group relationships between (a) the endemic viviparids of Lake Malawi and populations from the Middle Congo as well as between (b) the Victoria region and the Okavango/ Upper Zambezi area. Conclusions: The current study testifies to repeated disruptions of the distribution of the Viviparidae during the formation of the East African Rift System, and to a central role of the system for the distribution of the continent’s freshwater fauna during the late Cenozoic. By integrating our results with previous findings on palaeohydrographical connections, we provide a spatially and temporarily explicit model of historical freshwater biogeography in tropical Africa. Finally, we review similarities and differences in patterns of vertebrate and invertebrate dispersal. Amongst others we argue that the closest relatives of present day viviparids in Lake Malawi are living in the Middle Congo River, thus shedding new light on the origin of the endemic fauna of this rift lake. Keywords: Freshwater biogeography, East African Rift System, Congo, Lake Malawi, Lake Tanganyika, Zambezi

Background The three largest drainage systems in present-day Cen- The formation of the East African Rift System (EARS) tral Africa are the Congo-, the Nile-, and the Zambezi played a decisive role in the evolution of Africa’s tropical system (Figure 1). The Congo River covers an area of ~3.7 fauna and flora. The climatic, geological, and hydrolo- million square kilometres and has a length of ~4100 ki- gical consequences of the rifting processes are critical to lometres [2]. It drains part of the EARS (including understand the emergence of the endemic species of the Lake Tanganyika) and borders to the Nile system in the savannahs and the Great Lakes as well as the origin of northeast. The Nile system receives water from Lake humankind. Rifting created, disrupted, redirected, and Albert and Lake Victoria and drains a region of ~3.2 connected major freshwater systems [1]. million square kilometres for almost 7000 kilometres northwards to the Mediterranean Sea. To the south, the

* Correspondence: [email protected] Congo system borders to the Zambezi drainage system, 1Division of Genetics and Physiology, Department of Biology, University of which runs ~2600 kilometres, covers an area of 1.3 million Turku, Itäinen Pitkäkatu 4, 20014 Turku, Finland square kilometres [2,3], and includes the southernmost 2Department of Ecology & Systematics, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, Giessen 35392, Germany of the Great Lakes, Malawi. The central location of the Full list of author information is available at the end of the article EARS to these river systems bears witness to the rift’s

© 2014 Schultheiß et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Schultheiß et al. BMC Evolutionary Biology 2014, 14:42 Page 2 of 12 http://www.biomedcentral.com/1471-2148/14/42

Nile drainage system ngo River im Co ruw i A Congo drainage system Lake Muhazi

Lake Victoria

Ka Lake Tanganyika sa i Lake Mweru N

Okavango Lake Malawi drainage Lake Bangweulu system

Zambezi drainage U system .

Z mbezi O a Za k m a b v e a z n i sample locality g o lake river course Chobe drainage system border Lake 0 100 200 500 km Palaeo-Makgadikgadi

Figure 1 Map of tropical Africa with major drainage systems. The map shows the major drainage systems, framed with bold grey lines, and the sampling points of the study. Lakes and rivers referred to in the text are labelled accordingly; the Upper Zambezi is abbreviated as ‘U. Zambezi’. The dashed outline indicates the maximum expansion of Lake Palaeo-Makgadikgadi, modified after Riedel et al. [4]. Locality details for each sampling point are given in Table 1. importance in shaping drainage pattern of tropical Africa, centre of the continent and palaeohydrological research but their history since the Mid-Miocene is only partly re- indicates that the river and its tributaries have a highly solved and arguably complex [2,3]. vicissitudinous drainage history. Temporal connections Most suggestions of palaeohydrological connections in existed to the present day Lake Victoria area [14,15], Lake tropical Africa are based on circumstantial evidence in Turkana [16,17], the Upper Zambezi area [18,19] and the combination with general models of rifting and uplifting Indian Ocean [19], although the latter connection is con- [2] rather than on geomorphological evidence like in other troversial [1]. Hence, the analysis of Congolese samples is parts of the continent where palaeochannels can be traced a prerequisite for our understanding of the evolution and over considerable areas and are reasonably well dated, e.g. distribution of the freshwater fauna of tropical Africa— [5-7]. The consequential lack of a coherent synthesis or including the origins of the endemic radiations in the rift- a model of hydrographical connections since the Mid- lakes Malawi and Tanganyika. Miocene severely hampers our understanding of historical Here we study the historical biogeography of the fresh- biogeography in tropical Africa. This is further compli- water gastropod family Viviparidae, a taxon widely dis- cated by a scarcity of samples from the Congo River sys- tributed in the lotic and lentic environments of tropical tem, e.g. [8-11], but see Goodier et al. [12] and Day et al. Africa [20]. This family is of particular interest to studies [13]. The Congo is the largest freshwater system in the of historical freshwater biogeography because its dispersal Schultheiß et al. BMC Evolutionary Biology 2014, 14:42 Page 3 of 12 http://www.biomedcentral.com/1471-2148/14/42

is mostly limited to habitats with existing freshwater con- nodes were collapsed into a polytomy in Figure 2. The nections [21], rendering it an ideal tracer of past and number of parsimony informative sites and effective sam- present hydrographic connections. The ovoviviparous and pling sizes are provided in Additional file 1 for each frag- dioecious reproduction mode of viviparids minimizes op- ment separately. portunities for successful vector-mediated migration and a The phylogenetic structure revealed by Figure 2 also subsequent establishment of a viable population [22,23]. indicates that morphological identifications and geneal- Accordingly, viviparid fossils are conspicuously absent ogy do not match each other perfectly. A main concern from African water bodies that were never hydrographic- is the systematic position of Bellamya capillata, which ally connected to a source, e.g. crater- and oasis lakes [21]. has been traditionally reported from many localities in We obtained samples from the major drainage systems Africa south of the equator [20]. The type locality for in the region, i.e. that of the Nile, the Okavango, the this taxon is Lake Malawi, and all other localities from Zambezi as well as the Upper and Middle Congo. Using which the species was identified, are in fact inhabited by Bayesian multi-locus phylogenetic analyses and a fossil- populations that are deeply divergent from B. capillata calibrated, relaxed molecular clock we studied the diver- in Lake Malawi. Similar issues exist for other Bellamya sification and distribution of viviparids in Africa. In light species (see Discussion). We thus tentatively name spec- of the impact of the evolution of the EARS on African imens with an ambiguous status in current systematics hydrographic systems and the dependence of our model using ‘confer’ (cf.) to emphasize that taxonomic revisions organism on these systems to disperse, we expect a deep are required. phylogenetic separation between viviparid species east and west of the western branch of the rift system. We Molecular clock estimates furthermore hypothesize this separation to temporarily We focus on estimates for the ages of the following coincide with the disruption of freshwater migration routes three nodes in Clade I (Figure 2): Split A—most recent across the evolving rift. common ancestor (mrca) of the entire clade: 10.47 mil- lion years (My) (95% confidence interval [CI]: 6.69-15.04 Results My); Split B—the split between the Victoria- and the Phylogenetic analyses Okavango group: 6.32 My (CI: 3.58-9.96 My); Split C— The Bayesian analyses of the African Viviparidae yiel- the split between the Congo- and the Malawi group: ded one well-supported Neothauma group from Lake 4.52 My (CI: 2.32-7.70 My). The age of the mrca of the Tanganyika (Figure 2; Bayesian posterior probabilities Southern group in Clade II is 1.916 My (CI: 0.88-3.31 [BPP] 1.0) and two Bellamya clades comprising speci- My). Note that the here estimated mean substitution mens from the sampled major drainage systems of tropical rate of the COI fragment is 0.93% per My (CI: 0.68-1.18) Africa (Clades I and II in Figure 2; BPP 0.88 and 1.0). Sep- and therewith very similar to the trait-specific COI Pro- arate analyses of the nuclear and the mitochondrial data- tostomia clock rate for the HKY model suggested by set revealed no phylogenetic incongruences (Additional Wilke et al. [24] for dioeceous tropical and subtropical file 1). Clade I contains four well-supported, reciprocally taxa with a generation time of 1 year and a body size of monophyletic groups of specimens from (i) lakes Albert approximately 2–50 mm (mean rate: 1.24% per My; CI: and Victoria and the Nile River (‘Victoria group’;darkred 1.02-1.46). Substitution rates are provided in Additional in Figure 2; BPP 1.0); (ii) the Middle Congo River (‘Congo file 1 for each fragment. group’; light green in Figure 2; BPP 1.0); (iii) the rivers Chobe/Cuando and Okavango (‘Okavango group’;light Discussion red in Figure 2; BPP 1.0) and (iv) the Malawi Basin The phylogenetic reconstruction reveals two, geograph- (‘Malawi group’; dark green in Figure 2; BPP 1.0). Within ically highly disjunct sister-group relationships in Clade Clade I the Congo group is sister to the Malawi group I (Figures 2 and 3-4): the Congo-Malawi-cluster and the whereas the Victoria group is sister to the Okavango Victoria-Okavango-cluster. The groups within each clus- group (both sister-group relationships are supported with ter are separated by at least one water divide. This con- BPP 1.0). Clade II consists of a Northern group with spe- spicuous spatial pattern stands in sharp contrast to the cimens from lakes Mweru and Bangweulu (highlighted in initially stated expectation that species east of the western blue in Figure 2) and from the Northern Kafue (BPP 0.98) branch of the EARS are closer related to each other than as well as a Southern group with specimens from the to species west of it, and vice versa. Vector-mediated dis- Upper Zambezi, the Chobe, and the Southern Kafue persal across water divides is an unlikely explanation for (BPP 1.0). Due to lack of support in the two deepest the observed biogeographical pattern for reasons men- nodes of the phylogeny (BPP < 0.7) we can make no tioned earlier. Moreover, given the wide spatial overlap of conclusive statement as to the monophyly of either the potential dispersal corridors of the two clusters, we the African Bellamya or the African Viviparidae. These would expect to observe population exchange between Schultheiß et al. BMC Evolutionary Biology 2014, 14:42 Page 4 of 12 http://www.biomedcentral.com/1471-2148/14/42

MiocenePliocene Quaternary Idiopoma sp. Idiopoma sp. Out. Anulotaia sp. Out. Trochotaia sp. 1.00 N. tanganyicense GB N. tanganyicense 7032 N. N. tanganyicense 7033 N. B. pagodiformis 9477 B. pagodiformis 9478 Clade II B. cf. capillata 14830 0.98 B. cf. monardi 16995 B. cf. capillata 9474 B. capillata 1.00 cf. 9473 B. cf. monardi 16987 B. crawshayi BcrGB01 B. crawshayi BcrGB02 B. cf. capillata 17053 1.00 B. cf. capillata 17539 B. cf. capillata 16569 B. cf. capillata 17545 B. cf. capillata 17544 1.00 B. cf. capillata 17549 Bellamya sp. 9494 Bellamya sp. 14840 B. cf. capillata 16564

B. cf. capillata 16567 Southern group Northern group B. cf. capillata 16566 B. capillata 10886 1.00 B. capillata 10885 Clade I B. robertsoni 6596 B. capillata 8983

B. robertsoni group 1.00 6597 Malawi B. jeffreysi 6608 B. cf. capillata 16986 1.00 Bellamya B. cf. capillata 16985 Bellamya B. cf. capillata 16984 group Split C B. cf. capillata 16983 Congo 0.88 1.00 B. monardi 9489 . B. cf. capillata 17554 Okav B. monardi 9483 group B. trochlearis 15494 B. unicolor 15707 1.00 B. unicolor 15733 Split A B. trochlearis 15193 B. rubicunda 6615 B. unicolor 15746 B. rubicunda 15312 1.00 B. cf. unicolor 15745 Split B B. rubicunda 15482 B. unicolor 15165 B. rubicunda 15483 B. unicolor 15476 B. unicolor 15495 Victoria group B. unicolor 15261 B. trochlearis 15192 B. costulata 15686 B. jucunda 15321

15 12.5 10 7.5 5.0 2.5 0 Ma Figure 2 Multi-locus molecular phylogeny of the African Viviparidae. The phylogeny comprises the genera Bellamya and Neothauma (abbreviated as ‘N.’). The phylogeny was dated using a fossil-calibrated uncorrelated lognormal clock model. Clade I consists of four colour-coded, geographically distinct subgroups from the Middle Congo, Lake Malawi, the Lake Victoria area and the Okavango area. Colours correspond to those in Figure 3. Clade II consists of a Northern group with specimens from lakes Mweru and Bangweulu (highlighted in blue) and from the Northern Kafue as well as a Southern group with specimens from the Upper Zambezi, the Chobe, and the Southern Kafue. Confidence intervals (95%) for age estimates of selected nodes are indicated by grey bars; Bayesian posterior probabilities are given for selected nodes; ‘Out.’ indicates the outgroup. Locality details are provided in Table 1. taxa from e.g. the Middle Congo and the Nile, or be- Historical biogeography of Bellamya in tropical Africa tween the Malawi- and the Zambezi/Okavango group, Changes in the structural geology of East Africa associ- because the involved vectors would have been able to ated with uplifting and rifting [25] as well as pronounced operate in the entire area. Yet the reciprocal monophyletic moisture cycles over the past 5 My—mainly governed by clades within our phylogeny argue clearly against such an orbitally-induced insolation at low altitudes [26,27]— exchange. have created a dynamic setting for palaeohydrographical In order to explain our findings we integrated our phy- connections and, hence, organismal dispersal. Against logenetic information with literature data on palaeohydro- this background, we propose the following biogeogra- graphical connections to provide a synthesized, spatially phical model, which explains the two, spatially disjunct and temporarily explicit model of historical freshwater sister-taxon relationships as a consequence of succes- biogeography in tropical Africa. Subsequently, we review sive changes in hydrographical connections between recently published studies on African fish biogeography the Congo River system and its neighbouring water and evaluate similarities and dissimilarities of vertebrate systems (stages 1–4 in Figure 3; refer to Figure 1 for and invertebrate dispersal patterns. names of water bodies): Schultheiß et al. BMC Evolutionary Biology 2014, 14:42 Page 5 of 12 http://www.biomedcentral.com/1471-2148/14/42

1 2

Clade I

Clade II Clade II Split A Clade I Clade I

3 4

N ? ? modern lake modern river course ? ? modern drainage system border migration border Clade II Clade II drainage direction ceased connection Split C Split B 0 500 1000 2000 km Clade I Clade I

Figure 3 Biogeographical model for Bellamya. The model comprises four progressive stages covering roughly (1) Middle Miocene, (2) Late Miocene, (3) Pliocene and (4) Pliocene/Early Pleistocene timeframes. The map shows the current borders and courses of the drainage systems and rivers as well as the current sizes of the lakes. Inset phylogenies track the diversification of Clade I: colours correspond to geographic ranges and the phylogenetic groups in Figure 2. The borders of the suggested ranges are approximations; detailed descriptions of the individual stages are given in the text. Question marks within the dashed areas in stages 3 and 4 indicate potential remnant populations of the clusters from stage 2.

(1) The earliest African viviparid fossils were found in Malawi-cluster inhabited the Upper Congo tropical regions [15] (Additional file 2). We thus catchment including the Chambeshi system— assume that this region is the geographical origin of which drained eastward into the Rufiji system an ancestral population of Clade I in the Middle/ until the Pliocene [19]—, the emerging Lake Malawi, Late Miocene (~12 Ma; Figure 3-1). and perhaps Palaeolake Rukwa [32] (Figure 3-2). (2) Subsequently this ancestral lineage split into the At that time no hydrographical connection existed Congo-Malawi- and the Victoria-Okavango-cluster betweentheUpperandtheMiddleZambezi (Split A in Figures 2 and 3-2; mean age ~10.5 My). [28,33,34] and there was no faunal exchange In the Late Miocene/Early Pliocene the waters of between the Victoria-Okavango-cluster and the the present day Lake Victoria region drained via a Congo-Malawi-cluster. precursor of the Aruwimi River into the Congo (3) In the Late Miocene/Early Pliocene the Okavango [14,15]. A contemporaneous connection from the group started to separate from the Victoria group Congo system to the headwaters of the Upper (Split B in Figures 2 and 3-3; mean age ~6.3 My). Zambezi was suggested via the [19,28,29], During a wet phase in East Africa around 3.0-2.8 Ma which served as a dispersal route for various fish taxa faunal elements of Congolese origin (e.g. molluscs [11,12,30,31]. This persistent freshwater connection and sponges) colonised the Turkana Basin for the last likely also enabled viviparid migration between the time and all subsequent invasions into the basin were present day Lake Victoria region and the Upper of Nilotic origin [16,17]. This data indicates that the Zambezi/Okavango watershed (Figure 3-2). We connection of the Victoria region to the Central postulate that contemporaneously the Congo- Congo ceased, at latest with the closure of the Beni Schultheiß et al. BMC Evolutionary Biology 2014, 14:42 Page 6 of 12 http://www.biomedcentral.com/1471-2148/14/42

Gap in the Early Pleistocene [15]butperhapsearlier that the separation of the Congo-Malawi-cluster (Split C [1]; a similar timeframe is assumed for the disruption in Figures 2 and 3-4) was a consequence of the disruption of the Okavango/Upper Zambezi-Congo connection of hydrological connections and therewith migration [3,28,35]. We argue that the final separation of the routes (e.g. due to rifting processes) rather than of dis- two groups was ultimately governed by these events persal from the Congo River system into Lake Malawi (Split B in Figure 2) but we acknowledge that the or vice versa. Dispersal would most likely have resulted confidence interval for Split B also allows for an in a pattern where the sink population is nested within earlier separation. Around this time, the ancestors of the paraphyletic source population instead of the ob- the current Congo-Malawi groups inhabited an served reciprocal and strict regional monophyly. The area encompassing the Upper Congo system, the Bellamya of the lakes Mweru and Bangweulu, which Chambeshi system, and young Lake Malawi, most re-colonized these water bodies from the Zambezi sys- likely using the northwest corner of the lake’s basin tem, illustrate such a case of dispersal (Figure 2). We as gateway [3,19](Figure3-3). hence also conclude that the general dominance of re- (4) Subsequently the connection between Lake Malawi gionally confined monophyletic clusters in our dataset and the Chambeshi/Upper Congo system ceased, points toward a prevalence of vicariance over dispersal which separated the Congolese from the Malawian processes in the historical biogeography of the taxon— Bellamya (Split C in Figures 2 and 3-4; mean in contrast to, e.g., some fish taxa (see below). age ~4.5 My). This faunal disruption may have been Viviparid fossils with an age of up to four million years a direct consequence of rifting in the Mweru-Mweru are reported from the Chiwondo Beds in the Northwest Wantipa fault zone and in the northern part of the of the Malawi Basin [46]. Because the mean age of the Malawi Rift [36-38]. The extant viviparid fauna of the split between the Malawi and the Congo group is of a lakes Mweru and Bangweulu is however no remnant similar age, our results do neither reject the possibility of this former Congo-Malawi-cluster (Figure 2). In of an uninterrupted evolutionary Bellamya lineage in fact, age and phylogenetic position of the Mweru/ Lake Malawi (the founder-flush model in Schultheiß Bangweulu viviparids suggest a re-colonization of the et al. [47,48]) nor the possibility that the lake was re- two lakes from the Zambezi via the Kafue River [39], colonized after its Pliocene fauna went extinct, e.g. during perhaps after a climatically-induced Pleistocene severe lake level low stands [41]. In any case, the onset of ecosystem crisis comparable to those in nearby molecular diversification of the modern endemic Malawi Lake Malawi in the east [40-42]andLakePalaeo- group started considerably later (1.39 Ma; CI: 0.56-2.61 Makgadikgadi to the west [43]. Moreover, the My) than the deposition of the Plio-Pleistocene Chiwondo polyphyletic pattern of the Mweru/Bangweulu Bed fossils. Thus it has paralleled the diversification of the viviparids indicates that the two lakes may have endemic Lanistes taxa of Lake Malawi [48]. been colonized multiple times from the Zambezi River system (Figures 2 and 3-4). In the Late Pliocene/Early Pleistocene the Upper Zambezi Lake Tanganyika was captured by the Middle Zambezi [28,34], Although Neothauma is currently endemic to Lake which enabled secondary contact between the Tanganyika, this has not been the case from the Late Okavango group and Clade II (Figures 2 and 3-4). Miocene to the Early Pleistocene when the ra- The observable geographical overlap between the diated in Palaeolake Obweruka [15]. This palaeolake two groups coincides with the area of Lake Palaeo- occupied the Lake Albert-Edward region and was similar Makgadikgadi, which existed in consecutive phases in size and depth to Lake Tanganyika prior to the uplift of overtheMiddleandLatePleistocene[43-45] the Rwenzori Mountains ~2.3 Ma [14,49]. The molecular (Figures 1 and 3-4). This lake may have stirred phylogeny indicates that the Neothauma lineage from populations of initially different geographic origin Lake Tanganyika evolved fairly isolated from the African (e.g. the Okavango group and Clade II) and, Bellamya (Figure 2). A similar spatial, temporal, and gen- hence, superimposed previous spatial patterns etic distinctness is reported for some fish taxa of Lake resulting in the current overlapping distributions Tanganyika [31,50,51]. It appears reasonable to assume of Bellamya groups. that the lake served as a refuge and harbours the sole survivors of the formerly more widely distributed and Lake Malawi morphologically diverse genus Neothauma.Moreover, The phylogeny presented here reveals an unanticipated the isolated evolutionary history of Lake Tanganyika’s sister relationship of Lake Malawi’sextantBellamya viviparid fauna implies that for the timeframe and mi- fauna to that of the Middle Congo. In light of the well- gration events discussed in this paper the lake served supported reciprocal monophyly of both groups we argue neither as a gateway nor as a dispersal route. Schultheiß et al. BMC Evolutionary Biology 2014, 14:42 Page 7 of 12 http://www.biomedcentral.com/1471-2148/14/42

Comparative biogeography of afrotropical freshwater taxa the Luangwa to the Upper Zambezi and the Okavango This paper is to our knowledge the first invertebrate [12], crossing the young rift at the narrow Rukwa-Rungwe study investigating patterns of historical freshwater bio- region prior to the uplift of the Ufipa plateau [54]. A simi- geography in tropical Africa using molecular data. Against lar corridor over the rift appears to have been feasible for the background of recent studies on African fish biogeog- mastacembelid eels [51] and, in fact, also for viviparids: raphy we here review patterns of vertebrate and inver- The Congo-Malawi-cluster would likewise have occupied tebrate biogeography with a strong focus on dispersal the Rukwa-Rungwe region (green area in Figure 3-2). It abilities, hybridization, and migration routes. would hence be highly improbable that members of the A striking difference between viviparid and fish pat- Victoria-Okavango cluster migrated contemporaneously terns lies in the phylogenetic relationships of taxa from from the Victoria region through this corridor across the the modern Lake Victoria region: fish specimens from Rukwa-Rungwe area into the Upper Zambezi/Okavango. the Lake Victoria region cluster generally with other East This would imply that members of this cluster migrated African species, e.g. [31,50,52,53]. In contrast, the sister- along the same hydrographical system as the members of taxon relationship between viviparids from the Lake the Congo-Malawi-cluster, yet without disrupting the here Victoria region and those from the Okavango/Upper revealed biogeographical pattern. Consequently, we sug- Zambezi is to date unique among the afrotropical fresh- gest the existence of a second east–west migration corri- water fauna. This pattern is remarkable because connec- dor (red area in Figure 3-2) connecting the Victoria region ting these two drainage systems requires crossing two with the Okavango/Upper Zambezi area. divides: (A) the Congo/Upper Zambezi water divide via a With the exception of some branches in Clade II, our precursor of the Kasai River and (B) crossing the emerging phylogeny generally features strict regional monophyly— western branch of the EARS. To provide a hypothesis as a pattern often absent in biogeographical studies of fish to the cause of the difference of biogeographical patterns in tropical Africa, e.g. [11,12,55]. This absence is gener- between fish and viviparid taxa, we suggest that the rifting ally attributed to hybridization and active, long distance process was the vicariance event that caused the main dispersal, e.g. [8,11,12,31,52], which hence appear to have phylogenetic separation of fish taxa into species east and played only a minor role in the historical biogeography of west of the western branch of the EARS. At this stage, viviparids. Therewith, superimposition of underlying dis- however, the afrotropical Viviparidae were already sepa- tributional patterns by recent active dispersal or hybridi- rated into the Congo-Malawi cluster and the Victoria- zation seems negligible for this gastropod group, which in Okavango cluster (in the aftermath of Split A; Figures 2 turn facilitates the reconstruction of original migration and 3). We argue that this initial Split A was invoked by a corridors. migration barrier—perhaps in the Congo Basin—that The decisive phylogenetic pattern notwithstanding, fur- inhibited viviparid dispersal yet allowed fish dispersal. In- ther sampling is required to substantiate the proposed trinsic biological properties may create differential abilities model. In particular, more sampling from the central to overcome ecological, behavioural, and physical barriers Congo area is desirable despite logistic challenges, e.g., to [30], e.g. differences in mobility may cause differential mi- search for putative remnants of the Victoria-Okavango- gration through corridors which only exit during annual cluster (red dashed area in Figure 3-3). Furthermore, spec- flooding. For example, two major flooding events since imens are required from the Rufiji-Ruaha system to test 2010 promoted the migration of fish from the Okavango our hypotheses associated with the Malawi-Congo-cluster DeltaintotheformerlydryBotetiriverbed.However, (green dashed area in Figure 3-3). Eventually, more com- Bellamya—which lives in permanent overspills of the prehensive sampling of the Lower Zambezi may shed light such as the Thamalakane River—did on the origins and evolution of Clade II. It is noteworthy not migrate into the (FR pers. observa- that such an extension of the dataset will also enable a sys- tion, 2010, 2011, and 2013). tematic revision of the genus Bellamya, as our work re- Eventually, the rifting process separated the two vivi- ported considerable incongruences between traditional parid clusters into the four modern groups (i.e. the and the results of molecular phylogenetic ana- Congo-, Malawi, Okavango-, and Victoria group; Splits B lyses, e.g. [47,56]. and C) subsequently to Split A. Each of these groups is (much like in fish) confined to one side of the western Conclusions branch of the EARS. We have presented a first integrated model of historic This pattern and comparative biogeography sheds new freshwater invertebrate biogeography in tropical Africa. light on freshwater migration corridors over the emer- By comparing our results to fish data we demonstrate ging rift. African tigerfish, for example, were suggested the importance of broad geographical and taxonomic to have migrated during the Late Miocene along an east– sampling encompassing taxa with different dispersal abi- west corridor from the Tanzanian Rufiji-Ruaha system via lities and life history traits. Our analysis shows that the http://www.biomedcentral.com/1471-2148/14/42 Table 1 Locality details and NCBI GenBank accession numbers of the specimens analysed in this study Schultheiß DS Water body Country Longitude Latitude Species Sp. ID Voucher ID COI mtLSU ncLSU H3 Congo Lake Tanganyika Tanzania 29.69946 −4.78543 N. tanganyicense 7032* ZMB113503 HQ012716 HQ012683 HQ012706 ta.BCEouinr Biology Evolutionary BMC al. et Congo Lake Tanganyika Tanzania 29.69946 −4.78543 N. tanganyicense 7033* ZMB113504 HQ012717 HQ012707 Congo Lake Tanganyika Zambia n/a n/a N. tanganyicense GB# FJ405843 FJ405709 FJ405598 FJ405739 Congo Congo River Congo 22.66692 2.09519 B. cf. capillata 16983 ZMB 113784 JX489348 JX489282 Congo Congo River Congo 22.66347 2.09981 B. cf. capillata 16984 ZMB 113785 JX489349 JX489283 Congo Congo River Congo 22.66347 2.09981 B. cf. capillata 16985 ZMB 113786 JX489246 JX489315 JX489350 JX489284 Congo Congo River Congo 22.66347 2.09981 B. cf. capillata 16986 ZMB 113787 JX489247 JX489351 JX489285 Congo Lake Bangweulu Zambia 29.70750 −11.45268 B. cf. capillata 14830 ZMB 113788 JX489224 JX489295 JX489328

Congo Lake Mweru Zambia 29.04460 −9.04567 B. cf. capillata 9473* ZMB 113515 HQ012725 HQ012692 JX489326 JX489259 2014, Congo Lake Mweru Zambia 29.04460 −9.04567 B. cf. capillata 9474* ZMB 113512 HQ012722 HQ012690 JX489260 14

Congo Lake Mweru Zambia 28.73133 −9.34801 B. pagodiformis 9477* ZMB 113510 HQ012720 HQ012688 JX489261 :42 Congo Lake Mweru Zambia 28.73133 −9.34801 B. pagodiformis 9478* ZMB 113511 HQ012721 HQ012689 JX489327 Congo Lake Mweru Zambia n/a n/a B. crawshayi GB01# FJ405844 FJ405695 FJ405596 FJ405746 Congo Lake Mweru Zambia n/a n/a B. crawshayi GB02# FJ405867 FJ405710 FJ405591 Nile Nile River Uganda 33.15524 0.48544 B. unicolor 15165 ZMB 113789 JX489226 JX489297 JX489330 Nile Nile River Uganda 31.50487 2.45933 B. unicolor 15476 ZMB 113790 JX489232 JX489303 JX489336 JX489270 Nile Nile River Uganda 32.93648 1.09002 B. unicolor 15707 ZMB 113791 JX489238 JX489307 JX489341 JX489275 Nile Nile River Uganda 32.09473 1.69255 B. unicolor 15733 ZMB 113792 JX489239 JX489309 JX489342 JX489276 Nile Nile River Uganda 32.33107 2.12373 B. unicolor 15746 ZMB 113793 JX489241 JX489344 Nile Lake Albert Uganda 30.91564 1.44854 B. rubicunda 6615* ZMB 113501 HQ012711 HQ012681 HQ012705 Nile Lake Albert Uganda 31.10057 1.58649 B. rubicunda 15312 ZMB 113794 JX489230 JX489301 JX489334 JX489268 Nile Lake Albert Uganda 31.32021 1.81842 B. rubicunda 15482 ZMB 113795 JX489233 JX489304 JX489337 JX489271 Nile Lake Albert Uganda 31.32021 1.81842 B. rubicunda 15483 ZMB 113796 JX489234 JX489305 JX489338 JX489272 Nile Lake Muhazi Rwanda 30.47826 1.84843 B. cf. unicolor 15745 ZMB 113797 JX489240 JX489310 JX489343 JX489277 Nile Lake Victoria Uganda 32.49402 0.03892 B. jucunda 15321 ZMB 113798 JX489231 JX489302 JX489335 JX489269 Nile Lake Victoria Uganda 33.58264 0.16176 B. trochlearis 15192 ZMB 113799 JX489227 JX489298 JX489331 JX489265 Nile Lake Victoria Uganda 33.58264 0.16176 B. trochlearis 15193 ZMB 113800 JX489228 JX489299 JX489332 JX489266 Nile Lake Victoria Uganda 33.60258 0.14067 B. unicolor 15261 ZMB 113801 JX489229 JX489300 JX489333 JX489267 Nile Lake Victoria Uganda 32.49402 0.03892 B. trochlearis 15494 ZMB 113802 JX489235 JX489339 JX489273

Nile Lake Victoria Uganda 32.49402 0.03892 B. unicolor 15495 ZMB 113803 JX489236 JX489306 JX489274 12 of 8 Page Nile Lake Victoria Kenya 34.02031 0.1091 B. costulata 15686 ZMB 113804 JX489237 JX489307 JX489340 Okavango Namibia 21.58754 −18.12071 B. monardi 9489* ZMB 113507 HQ012800 HQ012686 HQ012709 JX489263 Zambezi Zambezi River Zambia 23.08752 −13.52859 B. cf. capillata 16564 ZMB 113805 JX489242 JX489311 JX489278 http://www.biomedcentral.com/1471-2148/14/42 Table 1 Locality details and NCBI GenBank accession numbers of the specimens analysed in this study (Continued) Schultheiß Zambezi Zambezi River Zambia 23.23285 −14.38287 B. cf. capillata 16566 ZMB 113806 JX489243 JX489312 JX489345 JX489279 −

Zambezi Zambezi River Zambia 23.23285 14.38287 B. cf. capillata 16567 ZMB 113807 JX489244 JX489313 JX489346 JX489280 Biology Evolutionary BMC al. et Zambezi Wanginga River Zambia 22.84252 −15.08967 B. cf. capillata 16569 ZMB 113808 JX489245 JX489314 JX489347 JX489281 Zambezi Kafue River Zambia 28.20085 −15.81976 Bellamya sp. 14840 ZMB 113809 JX489225 JX489296 JX489329 Zambezi Kafue River Zambia 26.04066 −14.81798 B. cf. monardi 16987 ZMB 113810 JX489248 JX489316 JX489352 JX489286 Zambezi Kafue River Zambia 26.10316 −14.70344 B. cf. monardi 16995 ZMB 113811 JX489249 JX489317 JX489353 JX489287 Zambezi Zambezi River Zambia 23.09447 −13.55640 B. cf. capillata 17053 ZMB 113812 JX489250 JX489318 JX489354 JX489288 Zambezi Zambezi tributary Zambia 22.95794 −15.19352 B. cf. capillata 17539 ZMB 113813 JX489251 JX489319 JX489355 JX489289 Zambezi Zambezi River Zambia 23.24116 −16.24204 B. cf. capillata 17544 ZMB 113814 JX489252 JX489320 JX489356 JX489290 2014, Zambezi Zambezi River Zambia 23.24116 −16.24204 B. cf. capillata 17545 ZMB 113815 JX489253 JX489321 JX489357 JX489291

Zambezi Zambezi, River Zambia 23.56652 −16.65075 B. cf. capillata 17549 ZMB 113816 JX489254 JX489322 JX489358 JX489292 14 Zambezi Zambezi River, Zambia 25.186540 −17.75630 B. cf. capillata 17554 ZMB 113817 JX489255 JX489359 JX489293 :42 Zambezi Cuando River Namibia 23.33815 −17.98192 Bellamya sp. 9494* ZMB 113518 HQ012728 HQ012695 JX489264 Zambezi Lake Kazuni Malawi 33.64470 −11.14642 B. capillata 10885* ZMB 113524 HQ012741 Zambezi Lake Kazuni Malawi 33.64470 −11.14642 B. capillata 10886* ZMB 113525 HQ012742 Zambezi Lake Malawi Malawi 34.93017 −14.08923 B. robertsoni 6597* ZMB 113584 HQ012797 HQ012704 JX489324 JX489257 Zambezi Lake Malawi Malawi 34.93017 −14.08923 B. robertsoni 6596* ZMB 113574 HQ012752 HQ012698 JX489323 JX489256 Zambezi Lake Malawi Malawi 34.29794 −12.88355 B. jeffreysi 6608* ZMB 113567 HQ012735 HQ012700 JX489325 JX489258 Zambezi Shire River Malawi 34.90498 −15.38884 B. capillata 8983* ZMB113545 HQ012734 HQ012699 HQ012711 Zambezi Chobe River 25.12985 −17.81595 B. monardi 9483* ZMB 113508 HQ012718 HQ012687 JX489262 Taxonomic remarks: Bellamya capillata is reported to be widely distributed in the Zambezi system [20] but previous studies showed that the species is endemic to Lake Malawi [47,56]. We thus named morphologically similar specimens from elsewhere B. cf. capillata as a taxonomic revision is beyond the scope of this paper. Abbreviations: DS—drainage system; Sp. ID—specimen identity number; n/a—data not available; *specimen from Schultheiß et al. [47]; #specimen from Sengupta et al. [56]; ZMB— collection of the Berlin Museum of Natural History. ae9o 12 of 9 Page Schultheiß et al. BMC Evolutionary Biology 2014, 14:42 Page 10 of 12 http://www.biomedcentral.com/1471-2148/14/42

biographical history of viviparids in tropical Africa is dri- and is ~19 My old [66]. To implement this calibration ven by consecutive vicariance events rather than by dis- point, we used a uniform prior over the root of the phy- persal and is closely linked to the formation of the EARS. logeny spanning 23–13 My to account for dating un- Moreover, we demonstrate that the Congo drainage sys- certainty and the various published hypotheses as to when tem plays a central role in the distribution of the conti- the Bellamyinae invaded Africa from Asia, e.g. [66-68]. nent’s freshwater fauna during the late Cenozoic. Our data The oldest Neothauma fossil (Neothauma hattinghi Van also sheds new light on the geographic origin of the en- Damme & Pickford, 1999) is reported from the Upper demic fauna of Lake Malawi and reveals its close phylo- Miocene Kakara Formation from the western branch genetic relationship to Congolese populations. We hope of the East African Rift System and is 10–11 My old that future work, with a further inclusion of invertebrate [15,69]. For this second calibration point we used a nor- taxa, will help to refine our understanding of biogeogra- mally distributed age prior (mean age: 10.5 My, standard phical patterns of tropical Africa’sfreshwaterfauna. deviation: 0.5 My) over the basal node leading to the monophyletic Neothauma clade (i.e. stem calibration Methods in BEAST 1.75). Uncorrelated lognormal relaxed clock Sampling and species identification models were chosen to account for lineage specific rate Specimens of Viviparidae were collected between 2006 heterogeneity within each partition; mixing of the Markov and 2012 in tropical Africa (Figure 1; Table 1). There are chains was monitored with Tracer 1.5 [70]. two extant genera of the family on the continent: the Ancestral area/state reconstructions as implemented widely distributed Bellamya and the monospecific Neo- in, e.g., Lagrange, BEAST and Mesquite are not feasible thauma, which is endemic to Lake Tanganyika [20]. Due with the present datasets for two reasons. First, the basal to the ambiguous phylogenetic relationship between the topology of the African viviparids is unresolved, whereas twogenera[47,56],wefurthermoreaddedthefollow- a dichotomy is needed to reconstruct the ancestral state ing closely related Asian viviparid genera to our data- and the dichotomy-enforcing algorithm of BEAST would set: Anulotaia, Idiopoma,andTrochotaia. Material was result in low node supports. Secondly, the four major identified before molecular analysis based on shell mor- groups of Clade I (Malawi, Congo, Okavango, and Victoria phology following the currently accepted taxonomy of group) form a dichotomous topology (Figure 2), resulting African Viviparidae, e.g. [20]. in non-informative ancestral state reconstructions for Split C (Malawi/Congo) and Split B (Okavango/Victoria), DNA extraction and amplification and an ambiguous state reconstruction for the most basal We used a CTAB protocol for DNA extraction [57] and Split A due to lacking information (unresolved topology) amplified four DNA fragments: the cytochrome oxidase of the most common recent ancestor. subunit I (COI) with universal [58] and specific primers [47], the mitochondrial large subunit of ribosomal RNA Availability of supporting data (LSU rRNA) [59], a fragment of the nuclear LSU rRNA The data sets supporting the results of this article are [60], and the nuclear histone 3 [61]. PCR conditions were available in the treeBASE repository, http://purl.org/ as given in [62] and sequencing was performed on an phylo/treebase/phylows/study/TB2:S15381. ABI3730XL sequencer (LGC Genomics Germany, Berlin). Sequences of all fragments were aligned unambiguously with ClustalW 1.4 [63] in BioEdit 7.1 [64] and checked Additional files by eye. Additional file 1: The supplementary file contains additional information on the phylogenetic analyses conducted during this Phylogenetic analyses and molecular clock estimations study. We furthermore provide the results of the separate analyses of We ran five independent Bayesian phylogenetic recon- nuclear and mitochondrial data. structions (partitioned into the four DNA fragments) in Additional file 2: The supplementary file contains additional information on the fossil record of the African Viviparidae and BEAST 1.75 [65] to ensure convergence on the same poster- the dating constrains employed in the phylogenetic analysis of ior probability distribution. An unlinked HKY + Γ substi- this study. tution model was utilized because more complex models showed indications of overfitting in preliminary runs; as Abbreviations tree prior we used a Yule model. In order to estimate EARS: East African Rift System; BPP: Bayesian posterior probabilities; divergence times we employed a fossil-based calibra- cf.: Confer; mrca: Most recent common ancestor; My: Million years; tion using the earliest bellamyinid viviparid from Africa CI: Confidence interval; COI: Cytochrome oxidase subunit I; LSU rRNA: Large subunit of ribosomal RNA. and the oldest Neothauma fossil (for an in-depth dis- cussion see Additional file 2). The oldest Bellamya occurs Competing interests in the Iriri Member of the Napak Formation at Napak The authors declare that they have no competing interests. Schultheiß et al. BMC Evolutionary Biology 2014, 14:42 Page 11 of 12 http://www.biomedcentral.com/1471-2148/14/42

Authors’ contributions 9. Wilson AB, Glaubrecht M, Meyer A: Ancient lakes as evolutionary RS analysed the data, developed the model and led the writing of the reservoirs: evidence from the thalassoid gastropods of Lake Tanganyika. manuscript. BVB developed the fossil framework of the study and contributed Proc R Soc Lond B 2004, 271:529–536. to the model building and writing. FR, TvR, and CA designed and coordinated 10. Schultheiß R, Ndeo OW, Malikwisha M, Marek C, Bößneck U, Albrecht C: the study; TvR and CA provided the DNA sequences. Material was collected by Freshwater molluscs of the Eastern Congo: notes on taxonomy, FR, BVB, and RS. All authors read and approved the final manuscript. biogeography and conservation. Afr Invertebr 2011, 52:265–284. 11. Day JJ, Bills R, Friel JP: Lacustrine radiations in African Synodontis catfish. J Evolution Biol 2009, 22:805–817. Accession numbers 12. Goodier SAM, Cotterill FPD, O’Ryan C, Skelton PH, de Wit MJ: Cryptic All DNA sequences used in this study are available from NCBI GenBank. diversity of African tigerfish (genus Hydrocynus) reveals Accession numbers are provided in Table 1. palaeogeographic signatures of linked Neogene geotectonic events. PLoS One 2011, 6:e28775. 13. Day JJ, Peart CR, Brown KJ, Friel JP, Bills R, Moritz T: Continental Acknowledgements diversification of an African catfish radiation (Mochokidae: Synodontis). We are grateful to Andrew Goudie, Jacek Stankiewicz, and Woody Cotterill Syst Biol 2013, 62:351–365. for an evaluation of the model. Daniel Engelhard, Thies Geertz, Andreas 14. Pickford M, Senut B, Hadoto D: Geology and Paleobiology of the Albertine Rift Schumann, Jean-Papy Mongindo, Ernest Tambwe, Martin Pickford, Ezra Valley, Uganda-Zaire. Volume I: Geology. 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